Various types of ribo- and deoxyribo-polynucleotide strands are known to form triple as well as double helical structures under physiological conditions of temperature and ionic strength. Recent findings have expanded the potential for complex-sequence polynucleotide single strands to form triple helices by associating with unique-sequence DNA double helices, and there is evidence that these structures can specifically repress the transcription of RNA and DNA templates in vitro. This project will further study three-stranded polynucleotide helices and investigate the potential for these structures to function as regulators of gene expression at the transcriptional level. Additional approaches for detection of in vitro formation of polynucleotide triplexes will be developed by the use of liquid chromatography, nuclease sensitivity studies, and structure-dependent quenching of fluorescent dyes. Stabilities and thermodynamic properties of the triplexes of varying size and sequence will be investigated through the use of unique-sequence DNA oligonucleotides, and UV thermodenaturation analyses. Formation and properties of various RNA/DNA hybrid triple helices will be studied, and in vitro RNA plymerase transcription systems will be developed to investigate the properties of these triplexes as inhibitors of gene expression.